AMP-activated protein kinase (AMPK) is considered as a cellular energy sensor that regulates glucose and lipid metabolism by phosphorylating key regulatory enzymes. Despite the major role of adipose tissue in regulating energy partitioning in the organism, the role of AMPK in this tissue has not been addressed. In the present study, we subjected AMPKα2 knockout (KO) mice to a high-fat diet to examine the effect of AMPK on adipose tissue formation. Compared with the wild type, AMPKα2 KO mice exhibited increased body weight and fat mass. The increase in adipose tissue mass was due to the enlargement of the preexisting adipocytes with increased lipid accumulation. However, we did not observe any changes in adipocyte marker expression, such as peroxisome proliferator–activated receptor-γ, CCAAT/enhancer-binding protein α (C/EBPα) and adipocyte fatty acid–binding protein (aFABP/aP2), or total cell number. Unlike impaired glucose homeostasis observed on normal diet feeding, when fed a high-fat diet AMPKα2 KO mice did not show differences in glucose tolerance and insulin sensitivity compared with wild-type mice. Our results suggest that the increase in lipid storage in adipose tissue in AMPKα2 KO mice may have protected these mice from further impairment of glucose homeostasis that normally accompanies high-fat feeding. Our study also demonstrates that lack of AMPKα2 subunit may be a factor contributing to the development of obesity.